Lysis and reverse transcription for mrna quantification

ABSTRACT

The present invention is directed to a method for performing RT-PCR for amplifying a target RNA comprising the steps of a) lysis of a cellular sample which is supposed to contain the target RNA with a lysis buffer comprising between 0.2 M and 1 M Guanidine Thiocyanate, b) diluting the sample to an extend such that Guanidine Thiocyanate is present in a concentration of about 30 to 50 mM, e) reverse transcribing in the presence of a mixture of first strand cDNA synthesis primers, the mixture consisting of oligo dT primers and random primers, and d) subjecting the sample to multiple cycles of thermocycling protocol and monitoring amplification of the first strand cDNA in real time, characterized in that steps a) to c) are done in the same vessel.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/797,878, filed on Jun. 10, 2010, now U.S. Pat. No. 9,200,313, whichis a continuation of U.S. application Ser. No. 12/582,737 filed Oct. 21,2009, which is a continuation of PCT/EP2008/003452 filed on Apr. 29,2008 and claims priority to EP07008961.0 filed on May 3, 2007.

FIELD OF THE INVENTION

The present invention provides a method for mRNA measurements. Usingquantitative PCR, together with optimized procedures for cellcollection, lysis and reverse transcription, the method allows the studyof transcript numbers, distributions, correlations, and gene inductioneven at the single cell level. cl BACKGROUND

Cells in a population are in many aspects unique in theircharacteristics, even in a seemingly homogenous culture or tissue. Geneexpression levels show large cell-cell variations, due to external(extrinsic) and internal (intrinsic) sources of factors. Likewise, whenexposed to identical stimuli, cells often behave stochastically. Thismeans that data obtained from a population of cells can not be assumedto reflect the behavior of the individual cell. It has been suggestedthat cells can respond to stimuli by bursts in transcriptional activityand operate as a binary switch: that is in an all-or-none fashion.

To determine whether two transcripts are expressed in a parallel(expression high at the same time) or anti-parallel (one is high whenthe other is low), transcription analysis at the level of the individualcell is required. When groups of cells are analyzed at the same time,important information is lost. For example, it is not possible todiscriminate between a small change in gene transcription occurring inevery cell as opposed to major changes in only a few cells. Furthermore,cell heterogeneity in tissues makes cell-type specific analysisdifficult. These issues are resolved by measurements in individualcells.

A typical eukaryotic cell contains about 25 pg of RNA of which less than2% is mRNA. This corresponds to a few hundred thousands of transcriptsof the ˜10,000 genes that are expressed in each cell at any particularpoint in time. Imaging techniques such as multiplex fluorescent insitu-hybridization (FISH) can monitor gene transcriptional activityspatially within a single cell by labeling of specific mRNAs and may beapplied to living cells to provide temporally resolved glimpses of thecomplexity of the transcription machinery. Protein levels in singlecells have been measured quantitatively in bacteria and yeast usingfluorescent reporter proteins. For a complete transcriptome analysis ofa single cell, microarrays, preceded by non-specific amplification ofcDNA, are used. The most widespread method for single cell mRNA analysisis reverse transcription polymerase chain reaction (RT-PCR), and therelated quantitative real-time RT-PCR (qRT-PCR). This technique offerssuperior sensitivity, accuracy, and dynamic range compared toalternative methods for mRNA measurements. The number of transcriptsthat can be readily analyzed in the single cell is small, butpre-amplification of cDNA vastly increases this number.

However, the protocols for single cell analysis that exist in the art sofar are useful only with respect to the detection of abundantlyexpressed targets. These methods do not provide the sensitivity requiredto detect target mRNAs that are expressed as a comparatively low level.

SUMMARY OF THE INVENTION

Thus, in a first aspect, the present invention is directed to a methodfor performing an RT-PCR. for amplifying a target RNA comprising thesteps of

-   a) in a sample vessel, lysis of a biological sample consisting of    only a few cells which is supposed to contain said target RNA with a    lysis buffer comprising between 0.05 M and 1 M Chaotropic agent-   b) in the same sample vessel, diluting said sample to an extend such    that Chaotropic agent is present during step c) in a concentration    of about 10 to 60 mM-   c) in the same sample vessel without any intermediate purification    step reverse transcribing said target RNA in the presence of a    mixture of first strand cDNA synthesis primers into a first strand    cDNA, said mixture consisting of primers hybridizing to a poly-A    sequence and/or random primers-   d) amplifying said first strand cDNA by means of subjecting said    sample to multiple cycles of a thermocycling protocol.

Preferably, amplification of said first strand cDNA during saidthermocycling protocol is monitored in real time.

This method is typically applicable if the sample comprises only alimited number of cells, i.e. not more that 1000 cells, and preferablyless than 100 cells. In particular, the method is applicable even if thesample comprises only less than 10 cells or a single cell.

Preferably, said chaotropic agent is Guanidine Thiocyanate.Alternatively said chaotropic agent may be come selected from a groupconsisting of Guanine Hydrochloride, Potassium Cyanate and Ammoniumsulphate.

Preferably, the lysis buffer comprises between about 0.2 and 0.5 MChaotropic agent.

Also preferably, during step c), i.e. during the reverse transcriptasereaction, Chaotropic agent is present in a concentration of about 10-60mM, more preferably between 30 and 50 mM and most preferably about 40mM.

Also preferably, step a) comprises the addition of a carbohydrate, whichis preferably a sugar or a dextran in order to avoid evaporation of lowsample volumes.

Optionally, the reverse transcriptase reaction may be performed in thepresence of 0.5 to 2% of a non ionic detergent, which is preferably NP40 (octylphenoxylpolyethoxylethanol). In one embodiment, such non ionicdetergent may already be added to the sample during step a).

In one embodiment, step a) is performed for at least 5 minutes atambient temperature or even below.

In another embodiment, the incubation of step a) may be performedbetween 55-85° C. in the presence of Proteinase K. Then, between step a)and step b) or step b) and step c), the sample may be incubated for atleast 5 minutes at a temperature between about 80° C. to 90° C. in orderto destroy any residual Proteinase K activity.

Also according to the present invention, step a) may be performed in thepresence of a double strand specific DNAse, preferably DNAse 1 or ShrimpNuclease. In case of using DNAse I, it is advantageous, if between stepa) and step b) or step b) and step c), the sample is incubated for atleast 5 minutes at a temperature between about 80° C. to 90° C.

Optionally, the sample is frozen at temperatures between about −20° C.and −80° C. immediately prior to step b). That means that afterfreezing, no additive as disclosed above is being added anymore prior tostep b).

According to the present invention it is highly preferable if saidmixture of (DNA synthesis primers comprises both: primers hybridizing toa poly-A sequence as well as random primers.

The random primer is usually a random hexamer primer. The primershybridizing to a poly-A sequence are usually oligo-dT primers orOligo-dU primers. In a preferred embodiment, said primers hybridizing toa poly-A sequence and random primers are present in equal amounts. Inanother preferred embodiment, which is compatible with the abovementioned one, said primers hybridizing to a poly-A sequence and randomprimers are present in concentrations between 1 μM and 5 μM each. Highlypreferred are concentrations of about 2.5 μM each.

In a very specific embodiment suitable for analyis of multicellularbiological samples, the method according the present invention comprisesthe steps of

-   a) lysis of a biological sample which is supposed to contain said    target RNA with a lysis buffer comprising between 0.2 M and 1 M    Guanidine Thiocyanate by means of-   incubation for at least 5 minutes at a temperature at ambient    temperature between 16° C. and 24° C. in the absence of proteinase K    or between 55-85° C. in the presence of Proteinase K-   incubation for about 2 to 10 minutes at 95° C.-   freezing said sample at −25° C.-   b) diluting said sample to an extent such that Guanidine Thiocyanate    is present in a concentration of about 20 to 60 mM.-   c) reverse transcribing in the presence of a mixture of first strand    cDNA synthesis primers, said mixture consisting of random sequence    primers and primers hybridizing to a poly-A sequence.-   d) subjecting said sample to multiple cycles of a thermocycling    protocol and monitoring amplification of said first strand cDNA in    real time.

In another very specific embodiment suitable for analyis of singlecells, the method according the present invention comprises the steps of

-   a) lysis of a single cell which is supposed to contain said target    RNA with a lysis buffer comprising between 0.2 M and 1 M Guanidine    Thiocyanate by means of-   incubation for at least 10 minutes at a temperature between at    ambient temperature between 16° C. and 24° C. in the absence of    proteinase K or between 55-85° C. in the presence of Proteinase K-   freezing said sample at −75° C. to −80° C.-   b) diluting said sample to an extend such that Guanidine Thiocyanate    is present in a concentration of about 30 to 50 mM.-   c) reverse transcribing in the presence of a mixture of first strand    cDNA synthesis primers, said mixture consisting of primers    hybridizing to a poly-A sequence and random primers-   d) subjecting said sample to multiple cycles of a thermocycling    protocol and monitoring amplification of said first strand cDNA in    real time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overview of single cell gene expression profiling using qRT-PCR.The alternatives and important issues for each step in the procedurethat were addressed in this study are shown in boxes. Optional steps areshown in dotted arrows.

Evaluation of lysis buffers. FIG. 2A Determination of lysis efficiency.Each bar indicates relative yield of Ins2 using a single pancreaticislet (˜2000 cells) as starting material. Each islet was treated withindicated concentrations of either NP-40, with and without proteinase K(Prot K), or guanidine thiocyanate (GTC). Only lysis with 0.5 M GIC hada significant effect compared to control conditions (p<0,001, n=3). FIG.2B Effect of lysis buffers on RT reaction yield. Identical amounts ofpurified islet total RNA was used as starting material. Relative yieldsof five genes were analysed: Ins1, Gcg, Sst, Gapdh and Rps29. Increasingconcentrations of guanidine thiocyanate was added to the RT reaction.There is a significant difference for all genes between control and 40mM or 120 mM (p<0.05) but riot 80 mM. Values are mean±SEM for 3 separateexperiments.

Optimization of the RT reaction. Four genes were measured: Ins1, Ins2,Rps29 and Hprt. FIG. 3A Determination of optimal RT primer concentrationusing either oligo(dT) or random hexamers. FIG. 3B Comparison of RTpriming strategies and temperature profiles. Identical amounts ofpurified total RNA from MIN6 cells was used as starting material.Relative RT reaction yields are shown for various primer combinations.2.0 μM of either oligo(dT) or random hexamers or both was used.Temperature profiles used are isothermal (black bars), gradient (whitebars) and cycled (grey bars). Values are mean±SEM for 3 separateexperiments.

Technical reproducibility of RT and qPCR. FIG. 4A Dilutions of purifiedtotal islet RNA, equivalent with the amount found in a single cell, wererun in triplicate RT and triplicate qPCR reactions. Standard deviation(SD) of measurements on three genes (Ins1, Ins2, and Gcg) is shown, withthe contribution from qPCR (black squares) and RT (red circles), The SDof qPCR triplicate reactions based on pre-determined amounts of purifiedPCR product of Ins1, Ins2, Gcg, Rps29 and Hprt are shown as reference(solid black line). The variability between these five assays is shownas (SD) error bars. FIG. 4B Single β-cells were measured with triplicateRT and duplicate qPCR reactions to visualize the variability in singlecell mRNA measurements. The Ins2 copy number refers to numbers ofmolecules in the RT reaction. Approximately 3% of the original cell wasanalyzed in each reaction.

Ins2 transcripts quantified in 126 β-cells from the Islets ofLangerhans. FIG. 5A The expression level of Ins2 for each β-cell,incubated in 3, 6, 10 or 20 mM glucose, as indicated. FIG. 5B Thehistogram shows that the expression levels of Ins2 are lognormallydistributed. Transcript levels are mean-centered for the four glucoseconcentrations.

FIG. 6: Genes' expressions in individual cells collected by FluorescenceActivated Cell Sorting (FACS), extracted using the disclosed invention,and measured with reverse transcription and qPCR. Beta actin cDNAmeasured in individual cells from which mRNA was extracted in 0.2 M GTC(top), 0.1 M GTC (middle) and in pure water (bottom).

FIG. 7: RRN18S cDNA measured in individual cells from which mRNA wasextracted in 0.2 M GTC (top), 0.1 M GTC (middle) and in pure water(bottom). X-axis shows the relative amount of cDNA present per cell inlogarithmic scale.

FIG. 8A and FIG. 8B: Mean and standard deviation of the relative amountof 18S, ACTB and GAPDH DNA determined by QPCR after treatment with thedisclosed invention. The studied RNA samples were spiked with genomicDNA, and treated with either DNase I (FIG. 8A) or shrimp nuclease (FIG.8B). Controls without nuclease treatment are presented, as well assample not treated with reverse transcriptase (No RT controls), whichonly contain genomic DNA.

FIG. 9: Eight single-cell samples analyzed in triplicate RT-reactionsfor insulin II expression. Four of the single cell samples wereextracted with lysis buffer containing 0.5% NP-40 and four with 0.5 MGTC according to the disclosed invention. Intra-assay variation for eachsample is shown.

FIG. 10A Aliquot extracted with the RNeasy Micro kit from Qiagen. MeanCt for HPV16E7 was 377>HPV 16E6 was not possible to detect. FIG. 10BAliquot extracted using the disclosed invention. Mean Ct for HPV16E6 was30.6 and mean CT for HPV16E7 was 29.2.

FIG. 11A and FIG. 11B: Extraction and analysis of Caski cells. QPCRanalysis of HPV16E6(FIG. 11A) and 18S cDNA (FIG. 11 B) +/−DNasetreatment +/− RT.

FIG. 12: Genes' expressions in pelleted THP1 monocytes prepared usingthe disclosed invention at different lysis temperatures, and measuredwith reverse transcription and qPCR. Y-axis shows the relative yield ofcDNA per sample.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, it is possible to perform a lysis ofeukaryotic cells or even prokaryotic cells in a certain reaction vesseland in the same reaction vessel performing a Reverse Transcriptasereaction in order to generate single stranded cDNA. Thus, the presentinvention more precisely is directed to a method for performing anRT-PCR for amplifying a target RNA comprising the steps of

-   a) lysis of a biological sample which is supposed to contain said    target RNA in a sample vessel with a lysis buffer comprising between    0.05 M and 1 M of a chaotropic agent-   b) diluting said sample to an extend such that said chaotropic agent    is present during step c) in a concentration of about 10 to 60 mM in    said sample vessel-   c) without any intermediate purification step reverse transcribing    said target RNA in the presence of a mixture of first strand cDNA    synthesis primers, said mixture consisting of primers hybridizing to    a poly-A sequence and/or random primers and/or target specific    primers in said sample vessel-   d) subjecting said sample to multiple cycles of a thermocycling    protocol and preferably monitoring amplification of said first    strand cDNA in real time.

In order to control the process of harvesting, cell lysis and reversetranscription, the samples may be spiked with a control RNA. The controlRNA preferably is an artificial RNA which during the step of reversetranscription is transcribed into a cDNA that can be discriminated fromthe RNA of the sample. In case of using specific primers for the ReverseTranscription step, the artificial RNA may be derived from in vitrotranscription of a genetically engineered DNA template that eithercomprises an insertion or only partially represents the target sequencewhich shall become analyzed.

The biological sample may contain either eukaryotic cells or prokaryoticcells which are in suspension. In case there are only a few eukaryoticcells such as a single cell, preferably less then 10 cells, morepreferably less than 100 cells m at least less than 1000 cells, thesuspension volume may also be very low. Preferably, the suspensionvolume is less then 20 μl and most preferably less than 5 μl.

Biological samples containing only a few cells may be generated forexample by means of dilution of a cell suspension culture. For thesecases, the lysis reagent according to the invention may be addeddirectly to the cell suspension as long as the lysis reagent is providedat least in a 5:1 V/V excess, or preferably in a 10:1 V/V excess.

Biological samples consisting of a small number of cells such as notmore than 100 cells, preferably not more than 10 cells and mostpreferably single cells only, may also be generated by means offluorescence activated cell sorting (FACS). In this case, the sortedcells may be directed into a well such as a well within a microliterplate, which already contains the lysis buffer according to theinvention.

However, the method according to the present invention is alsoapplicable for a larger number of cells that have been cultivated insuspension. Preferably, large cell numbers are first collected byappropriate means of centrifugation. Optionally said collectioncomprises a washing step in an appropriate buffer system such as a PBSbuffer. Subsequently the lysis buffer is added. For lysis of thecomplete sample, the sample may be shaken vigorously, for example bymeans of vortexing.

In one embodiment, step a) of the present invention is performed in thepresence of a non ionic detergent such as NP40. Alternatively, step a)comprises the addition of a carbohydrate, which is preferably a sugar ora dextran. Both components result in an effective avoidance ofevaporation effects, if samples with only small volumes need to beprocessed. If NP40 or another non ionic detergent is added, then theamount of detergent should be chosen in such a way that during step c)said detergent is present in a V/V amount of 0.5 to 2%.

In a preferred embodiment, the lysis buffer according to the presentinvention additionally contains polyinosinic acid. Advantageously, 0,2-5ng and most advantageously, 0,5-2 ng polyinosinic acid are contained in1 μl of lysis buffer.

Cell harvesting and lysis according to the present invention may beperformed at various different temperatures. In one embodiment, step a)of the inventive method is performed in the absence of Proteinase K forat least 5 minutes i.e. between 16° C. and 24° C. The maximum time whichis required for lysis under these circumstances is about 30 minutes.Similarly, step a) of the inventive method in the absence of ProteinaseK can be performed for at least 10 minutes even below ambienttemperature, but above 5° C. The maximum time which is required forlysis under these circumstances is about 60 minutes. These conditionsare very favorable for the avoidance of any evaporation effects, ifsamples with small volumes are processed. It also eliminates the needfor heating.

In an alternative embodiment of the present invention, step a) isperformed for at least 5 minutes at a temperature between about 55 to85° C. preferably in the presence of Proteinase K in concentrations ofabout 0,05 to 5 m ml and preferably 0,1-1 mg/ml. Optionally, saidProteinase K may be irreversibly inactivated by means of subsequentincubation either between step a) and step b) or between step b) andstep c) for at least 5 minutes but usually not more than 30 minutes at atemperature between about 80° C. to 90° C.

As according to the present invention, cell lysis and reversetranscription are performed in the same reaction vessel, it has beenproven to be highly advantageous if the genomic DNA that is contained inthe lysed cells can selectively be removed, while the cellular RNA ismaintained intact. The most effective possibility to achieve this effectis an enzymatic removal by means of including a DNAse digestion step.Thus, in one major embodiment of the present invention, step a) isperformed in the presence of a double strand specific DNAse. Preferably,such a DNAse is an exclusively double strand specific. DNAs such asDNAse I or Shrimp DNAse (USB, Cat No: 78314).

However, if during step d) the single stranded cDNA is further besubjected to a DNA Polymerase catalyzed amplification reaction such as aPCR reaction, it is highly advantageous to inactivate said DNAse priorto the amplification reaction. Thus, for inactivation of DNAse activity,in a specific embodiment the sample is incubated for at least 5 minutesbut not longer than 60 minutes between step a) and step b) or step b)and step c) at a temperature between about 80° C. to 90°. Alternatively,if the DNAse is Shrimp DNAse, the denaturation during the first cycle ofthe PCR reaction in step d) is usually sufficient.

All the different embodiments as outlined above have in common that celllysis, dilution, any addition of additives and the Reverse Transcriptasereaction are carried out in the same reaction vessel. Therefore, theinventive method is particularly useful for high throughput analyses ofmultiple samples within an automated process. In this context, themultiple reaction vessels may be arranged together in the form of amicrotiter plate as it is well known in the art. For example such amicro-titer plate may consist of 24, 96, 384, or 1536 separate reactionvessels arranged to standards that are established in the art. Then, thelysis reagent, the various additives and the reagents necessary forperforming a Reverse Transcriptase reaction can be added to the samplesby liquid handling roboting instruments.

Optimization of Conditions for Performing RT-PCR Analysis on Small CellNumbers

The method according to the present invention also permits geneexpression profiling in only a few cells and even in a single cell.Species of mRNA in single cells or a few cells are quantified byqRT-PCR. At a cell population scale, gene expression levels are commonlynormalized to reference genes [15]. The stochastic nature of singlecells makes this approach invalid, leaving absolute quantification asthe best option to compare transcript levels within and between cells.In order to improve the experimental protocols to optimize cell lysisand mRNA accessibility, the mRNA yield in the reverse transcriptionreaction and quality assessment were thus systematically analyzed. Themethod was demonstrated on single cells from the pancreatic islets ofLangerhans in mouse, revealing transcript copy numbers, co-regulation ofgene expression, and distribution of transcript expression levels.

Dispersed cells were collected with a glass capillary, emptied in lysisbuffer and analyzed with qRT-PCR. FIG. 1 outlines the experimentalprocedure. For quality assessment, an artificial RNA molecule based onthe cyclophilin. E (Ppie) gene was generated by in vitro transcription.An equal amount of Ppie RNA was added together with lysis buffer to allreaction tubes before the single cell was deposited into the tube. TheRNA could reduce the adsorption of the cell itself or single cell mRNAsto surfaces. Samples with deviant cycle of threshold (Ct) values for thePpie RNA spike may result from degradation by RNases. These samples werere-analyzed and, if the problem remained, excluded from furtheranalysis. The presence of inhibitors may reduce the cDNA yield.

Optimization of Lysis and Buffer

The purpose of the lysis buffer is to make the mRNA accessible for theRT enzyme. Two detergents were chosen for this task, a weak,non-chaotropic (Igepal CA-630, a.k.a. NP-40) and a strong, chaotropic(guanidine thiocyanate, GIC). The lysis efficiency and potentialinfluence on the downstream RT reaction was evaluated. Five differentlysis conditions were evaluated in terms of their ability to lyse onepancreatic islet (FIG. 2A). NP-40 had no effect compared to control(water) when used at concentrations of 0.5% or 4%. Proteinase K had noeffect when added in the presence of 0.5% NP-40. GTC based lysis bufferprovided efficient lysis of the islet using a concentration of 0.5 M andincreased the RNA yield 600-fold; an effect that was strongly diminishedat 4 M.

Then, the lysis buffers with respect to their effect on the RI-reactionwere compared. Low concentrations (0.1%) of NP-40, regardless ofaddition of proteinase K, did not have an effect compared to controlconditions. However, when used at a concentration of 1%, NP-40 resultedin small but significant improvement of RT-efficiency. In FIG. 2B, threeconcentrations of GTC (40 mkt, 80 mM, and 120 mM) were tested on fivegenes. The reaction efficiency was significantly improved (2-6 fold) forall tested genes using 40 mM GTC. By contrast, 80 mM GTC had no effectwhereas 120 mM GTC was in fact inhibitory. The addition of 0.5-1-5%2-mercapto ethanol did not improve the yield of the RT reaction eitheralone or in concert with GTC (data not shown). Formation of correctPCR-products was confirmed by agarose gel electrophoresis. Of the testeddetergents, GTC was chosen due to superior lysis ability and positiveeffect on the RT reaction at the concentration of 40 mM.

Optimization of RT Priming

Proper quantification of rare transcripts requires efficient cDNAgeneration by reverse transcription (RI). In FIG. 3A, priming of the RTreaction by random hexamers and oligo(dT) nucleotides were investigatedfor four genes: insulin 1 (Ins1), insulin 2 (Ins2), ribosomal proteinS29 (Rps29) and hypoxanthine guanine phosphoribosyl transferase (Hprt).The effect of RT primer concentration was analyzed (FIG. 3A). The RTefficiency generally increased with increasing primer concentration,although there were some differences between the, measured genes. 2.0 μMof either oligo(dT) or random hexamer priming result in a high cDNAyield. In FIG. 3B, the effect of combining RT-primers and temperatureprofiles was tested. Three different temperature profiles (isothermal,gradient and cycled temperature profiles) were evaluated in combinationwith random hexamer and oligo(dT) priming strategies. The combination ofboth priming methods were in all cases superior or equal to the singlebest priming method used. It could be hypothesized that the initiationof the RT reaction, at which stage the RT primer anneals to its targetmRNA molecule, is critical. A gradually increasing temperature(gradient) would allow low melting point RT primers to anneal to itstarget, while strong secondary structures denature in the later stagesof the incubation. A cycled temperature profile was also tested whichwas recently reported to increase the cDNA yield for quantification ofmiRNA [14]. However, there was no significant difference in yield orreproducibility between the tested temperature profiles. There was2-5-fold difference between worst and best primer/temperaturecombination. One can conclude that a combination of 2.0 μM oligo(dT) and2.0 μM random hexamers maximizes the yield of the RT reaction.

In addition to oligo(dT) and random hexamer priming, RT-priming withgene-specific primers (GSP) was tested. For some genes, concentrationdependent formation of non-specific products in the downstream PCR wasobserved. This effect was pronounced when using a mixture of differentGSPs. To determine whether this was an effect on the RT or the PCR, GSPswere added directly to the PCR. A total concentration >0.4 μM GSPs inthe PCR resulted in formation of erroneous PCR products. However,dilution of cDNA reverse transcribed with GSP did only partly remove theformation of unspecific products. Thus, high concentrations of GSPsaffect both RT and PCR reactions negatively.

Technical Reproducibility

The technical reproducibility of RT and qPCR is presented in FIG. 4A.The reproducibility of the RT and qPCR reaction, represented here bystandard deviation in Ct values (SDRT and SDqPCR respectively), areshown for a range of different initial copy numbers. In addition, SDqPCRwas calculated from dilution of purified PCR-product. There is nodifference between SDqPCR from cDNA or PCR-product, indicating that thetechnical reproducibility is intrinsic of the qPCR reaction itself andnot due to interfering factors from upstream reactions. All reactionsare highly reproducible down to ˜1000 copies (approx. Ct 28-30), andacceptable down to ˜100 copies. At <100 copies the variability in the RTand PCR reactions is a considerable obstacle for accurate quantificationof mRNA. FIG. 4B shows the technical variation in context of thebiological, cell-to-cell variation. Single cells from the islets ofLangerhans in mice were collected and analyzed in triplicate RTreactions and duplicate qPCR reactions. The technical variation is onpar with the one observed in FIG. 4A. Though the cell-to-cell variationfor the collected β-cells was relatively small, the technical variationis smaller, and allows absolute quantification with sufficient accuracyin the range of 100-200 molecules.

For analysis of gene expression profiling in single cells from theendocrine pancreas of the mouse, Ins1, Ins2, glucagon (Gcg), Rps29 andchromogranin B (Chgb) were measured in 158 cells collected from fourincubations with different glucose concentrations (3, 6, 10 and 20 mM).Islets of Langerhans in the pancreas are heterogeneous and contains1000-3000 cells comprising at least four endocrine cell types, whereinsulin-secreting β-cells and glucagon-producing α-cells are the mostabundant [18]. Based on presence of insulin or glucagon transcripts,these cells were distinguished as β-cells (26 cells, 83%), α-cells (25cells, 16%) or unknown (1 cell, 0.7%). Six samples were negative for allmeasured genes and they were categorized as technical failures (96%success rate). For all genes, lognormal distribution was confirmed andthe geometric means were calculated as shown in the following table:

TABLE 1 Statistical parameters describing gene expression in singlepancreatic α- and β- cells Geometric log₁₀ Geometric Shapiro Wilk GeneN¹ mean mean (SD) P-value² Ins2 124 8900 3.9 (0.5) 0.53 Ins1 100 31003.5 (0.5) 0.57 Gcg 25 19000 4.3 (0.3) 0.25 Rps29 102 230 2.4 (0.3) 0.98Chgb 59 82 1.9 (0.5) 0.67 ¹N is the number of cells expressing thetested gene. N_(tot) = 158. ²A high Shapiro-Wilk value reflects a goodfit, in this case to the lognormal distribution.

The data allowed for a correlation of expression levels in theindividual cells as it is shown in the following table. The Pearsoncorrelation factor for Ins1 and Ins2 was 0.59, which indicates singlecell correlation.

TABLE 2 Pearson correlation coefficients of expression levels in singlecells Ins2 Ins1 Gcg Rps29 Chgb Ins2 1 Ins1 0.59 1 Gcg N.A. N.A. 1 Rps290.18 0.19 −0.25 1 Chgb 0.08 0.14 0.06 0.31 1

All populations of cells showed a large heterogeneity in transcriptlevels. For example, of the 35 β-cells incubated in 20 mM glucose, thefour cells with highest expression account for 50% of the total Ins2mRNA (FIG. 5A). As indicated in studies on pooled cells, increasingglucose concentration had a stimulating effect on Ins 1, Ins2 and Chgband a slight negative effect on Gcg. A stronger effect of glucose oninsulin and glucagon in the cells with high expression levels wasobserved. Out of the 14 cells with the highest Ins2 levels (top 20%), 11were incubated in 20 mM glucose while three were in 3 mM glucose.Corresponding ratio between 20 and 3 mM glucose incubations were 12 to 1for Ins 1 and 0 to 2 for Gcg. Expression levels were normallydistributed for all genes at every glucose concentration.

Like any investigation of small and sensitive mechanisms, single celltranscription profiling requires careful preparations and highlyoptimized reaction conditions for a successful outcome. The results ofthe disclosed study allows definition of optimal conditions that willenable to experimenter to avoid pitfalls by using the right reagents atthe ideal concentrations.

While the effect on lysis is indisputable and expected—i.e. GTC wassuperior in lysing the islet—its effect on the RT reactions is morecomplex. While high concentrations (>100 mM) severely disturb thereaction, low concentrations (˜40 mM) has a favorable effect on the RTyield. GTC is a strong chaotropic detergent. The positive effect of GTCmay act by reducing secondary structures, thus allowing greater accessto the mRNA for primers and reverse transcriptase.

According to the present invention, it is preferred to use a combinationof random hexamer and oligo(dT) in the RT reaction irrespective of thetemperature profile used. In agreement with previous findings [16], theresults are highly gene dependent and thorough optimizations is neededfor highest possible RT efficiency. Priming of RT by gene-specificprimers (GSP) is occasionally used in mRNA quantification. There are twoexplanations to the formation of unspecific PCR products of cDNA primedwith GSPs: Firstly, the total primer concentration in the cDNA used inthe PCR can reach levels approaching that of the PCR primers. This couldinterfere with the amplification and generate unspecific products.Secondly, GSPs bind largely unspecific to the mRNA [16]. Thus, theresulting cDNA will be similar to that of random hexamer priming, butwith the GSP primer in the 3′ end.

In five measurements of a cell with 100 copies of a particulartranscript, the results will span approximately 10-40 copies perreaction (corresponding to 50-200 copies per cell). This spread ismostly due to variation in the qPCR, and it is in line with thebiological variation between cells. Duplicate or triplicate qPCRreactions will provide increased accuracy, and allow quantification oflower levels.

In addition, the reproducibility of results obtained with single cellpreparations in NP40 in milliQ water was compared with preparations in0.3 M GTC by means of calculating the standard deviations of 6 RT-PCRreactions each. For this experiment, each cell was divided into three RTreplicates and each of these were later divided into PCR duplicates.Hence, the standard deviation (SD) was based on six samples. The resultsshowed that the standard deviation was is substantially lower (andtherefore reproducibility higher) for cells prepared in 0.3 M GTC.

Summarizing, according to the method of the present invention, the cellsare deposited in the strong detergent guanidine thiocyanate, optionallytogether with an RNA spike for quality assessment. 30 to 50 mMconcentration of guanidine thiocyanate is a potent reverse transcriptionreaction enhancer. A combination of random hexamer and oligo(dT) primingensures a high cDNA yield.

EXAMPLES Example 1 Preparation and Culture of Cells

Pancreatic islets were prepared from healthy female National MaritimeResearch Institute (NMRI) mice aged 3-4 months (Bomholtgaard, Ry,Denmark) and fed a normal diet ad libitum. The mice were sacrificed bycervical dislocation, and pancreatic islets were isolated by collagenaseP digestion (Roche, Basel, Switzerland) followed by manual collection ofislets. All experimental procedures involving animals were approved bythe ethical committee of Lund University. To prepare dispersed singlecells the collected islets were gently shaken at low extracellularCa^(2r) concentration to dissolve the structure of the islet [21].Dispersed cells were plated on plastic 35 mm Petri dishes (Nunc,Roskilde, Denmark) in RPM! 1640 medium (SVA, Uppsala, Sweden)supplemented with 10% 100 U mL⁻¹ penicillin, and 10 μgmL⁻¹ streptomycin(all from invitrogen, Carlsbad, Calif., USA) in the presence of variousconcentrations of glucose (Sigma-Aldrich, St. Louis, Mo., USA). Thecells were maintained in culture 18-24 hours for the glucose stimulationexperiment and for 2-6 hours for other experiments. MIN6 cells werecultured in 5 mM glucose as previously described [2].

Example 2 Single Cell Collection

Attached dispersed cells were washed twice with a buffer containing 138mM NaCl, 5.6 mM KCI, 1.2 mM MgCl₂, 2.6 mM CaCl₂, 5 mM HEPES (pH 7.4 withNaOH) and 3-20 mM glucose (same glucose concentration as in culture) toremove dead and loose debris for cell collection with patch-clamppipettes. The dish, containing adhered cells and approximately 1 mLbuffer, was mounted in a standard inverted light-microscope (ZeissAxiovert 135, Oberkochen, Germany). Borosilicate glass capillaries(Hilgeriberg GmbH, Malsfeld, Germany) with outer diameter of 1.6 trimand wall thickness of 0.16 mm were pulled to pipettes using apatch-clamp pipette puller (Heka PIP5, Lambrecht, Germany). The diameterof the tip was approximately 10 μm on average, substantially wider thanstandard patch-clamp pipettes and large enough to allow passage of anintact cell. The glass pipette was mounted on a hydraulicmicromanipulator (Narishige, Tokyo, Japan) on the microscope. Bycontrolling the pressure inside the pipette it was possible to collectintact or nearly intact cells with minimum volume of extracellularsolution.

Example 3 Lysis and Purification

Islet lysis: Single pancreatic islets of roughly the same size and about1000 cells were placed in 10 μl of various lysis buffers. The detergentsNonidet P-40 (NP-40, a.k.a. Igepal CA-630, Sigma-Aldrich) and guanidinethiocyanate (GTC, Sigma-Aldrich) were used. Samples were incubated at60° C. or 80° C. for 15 minutes (60 QC for samples containing 0.4 mg/mlproteinase K (Invitrogen)) followed by 5 min incubation at 95° C. andfrozen at −25° C. for subsequent analysis. Samples were diluted 1:20prior reverse transcription to minimize possible inhibitory effects.

Single cell lysis: In single cell experiments, the glass pipettes wereemptied in 0.2 ml plastic tubes containing 2 μl of lysis solution. Theemptying required a custom-made device, consisting of a tube holderlined up with a coarse mieromanipulator on which the pipette wasmounted. The glass pipette was carefully flushed with lysis buffer a fewtimes to make sure the cell entered the tube. In most cases, the tip ofthe pipette was gently broken in the tube thereby facilitating theflushing of the pipette. Tubes were then immediately placed on a heatingblock with heated lid at 80 ° C. (for 5 minutes. Several compositions ofthe lysis buffers, containing either NP-40 or CITC, were evaluated.Unless indicated otherwise, the detergents were diluted in mQ purifiedwater, but occasionally in a buffer containing 50 mM Tris-Cl pH 8.0, 140mM NaCl, 1.5 mM MgCl₂ (all Sigma) was used. Following the heattreatment, the samples were immediately frozen on dry ice (−78° C) andstored at -80 ° C. for subsequent reverse transcription.

Total RNA extraction: Some optimization experiments were performed withtotal RNA from larger cell populations. Total RNA was purified withGenElute Mammalian Total RNA Kit (Sigma-Aldrich) and concentrations weremeasured with a NanoDrop ND-1000 spectrophotometer (NanodropTechnologies, Wilmington, Del., USA).

Example 4 In Vitro Transcription

To generate an artificial RNA control we used the T7 RNA Polymerase invitro transcription system (Takara, Shiga, Japan). A PCR assay forcyclophilin E (P)ie) was used as template for the in vitrotranscription. First, the Ppie PCR product was generated using the samesetup as for the real-time PCR assays, except that all fluorophores wereexcluded. The PCR product was purified using PCR purification kit(Qiagen, Hilden, Germany) and then amplified again in a new PCR reactionwith an extended forward PCR primer where the promoter sequence for T7RNA Polymerase was added. The final PCR product was purified as aboveand used in the in vitro transcription reaction, according to themanufacturer's instruction. The 20 μL reaction mix contained: 40 mMTris-HCl (pH 8.0), 8 mM MgCl₂, 2 mM spermidine 5 mM dithiothreitol (allTakara) 2 mM NTP (Invitrogen), 20 U RNaseOut (Invitrogen) and ˜40 ngtemplate DNA. The reaction -was incubated at 42° C. for 1 h.

Example 5 Reverse Transcription

The reverse transcriptase SuperScript III (Invitrogen) was usedthroughout the study [17]. 6.5 μL containing total RNA or lysed singlecells, 0.5 mM dNTP (Sigma-Aldrich), 2.5 μM oligo(dT) (Invitrogen), 2.5μM random hexamers (Invitrogen) and if indicated 0.5 μM of each genespecific primer (identical to reverse PCR primer, Invitrogen orMWG-Biotech, Ebersberg, Germany) were incubated at 65° C. for 5 min.Various combinations of RT-primers were used in this work andalternative strategies are described in the results. We then added 50 mMTris-HCI (pH 8.3), 75 mM KCI, 3 mM MgCl₂, 5 mM dithiothrettol, 20 URNaseOut and 100 U SuperScript III (all Invitrogen) to final volume of10 μL. Final reaction concentrations are shown. The temperature profilesused were: isothermal, 25° C. for 5 min, 50° C. for 45 min: gradient,25-40° C. for 1 min/° C., 41-65° C. for 5 min/° C.; cycled, 50 cycles at25° C. for 30 sec, 50° C. for 30 sec and 55 ° C. for 5 sec. Allreactions were terminated at 70° C. for 15 min, For calculations ofstandard deviation of the RT-qPCR reaction, triplicate RT and triplicateqPCR reactions on diluted purified total mouse islet RNA was used [16].

Example 6 Quantitative Real-Time PCR

Real-time PCR measurements were carried out on the ABI PRISM 7900HTSequence Detection System (Applied Biosystems, Foster City, Calif., USA)in 10 och 20 μL, reactions. The PCR mix contained: 10 mM Tris (pH 8.3),50 mM KCI 3 mM MgCl2, 0.3 mM dNTP, 1 U JumpStart Taq polymerase (allSigma-Aldrich), 0.5 x SYBR Green I (Invitrogen), x Reference Dye(Sigma-Aldrich) and 400 nM of each primer (MWG-Biotech). Formation ofexpected PCR products was confirmed by agarose gel electrophoresis (2%)for all assays, and melting curve analysis for all samples. Real-timePCR data analysis was performed as described, and PCR efficiencies werecalculated from dilution series of purified PCR-products (QIAquick PCRpurification kit, Qiagen) [12,19]. Absolute copy numbers of purified PCRproducts were calculated using the following molar absorptivity values(in Moles-1cm-1): dAMP, 15200; dTMP, 8400; dGMP, 12010; dCMP, 7050. A260was measured with the NanoDrop ND-1000 spectrophotometer.

Example 7 Analysis of individual cells sorted by Fluorescence ActivatedCell Sorting

Individual THP1 monocytes were sorted into 96-well plates usingFluorescence activated cell sorting (FACS). Each well contained 100 ngpolyinosinic acid, 500000 copies of an artificial RNA spike and either2μl 0.2 M GTC, 4 μl 0.1 M GTC or 10 μl H₂O. Reverse transcription (RT)was performed directly on the lysates in a 20 μl reaction volume (20 mMfinal GTC concentration per RT reaction) using the Transcriptor FirstStrand cDNA synthesis kit (Roche) with a blend of oligo(dT) and randomhexamers. Two mRNAs, RRN18S and ACTB, were quantified per cell with qPCRand SYBR Green I Chemistry in a LightCycler480 (Roche).

Example 8 Removal of Deoxyribonucleic Acid as Integrated Step in theDisclosed Invention.

RNA samples contaminated with DNA were generated by spiking 5 ng ofhuman total RNA (Roche) with 250 ng human genomic DNA, 2 ul of lysisbuffer (0.2 M GTC, 10 ng/ul poly(I)) was added to eight samples togetherwith either H₂₀ O (4 samples) or 1 U nuclease (either DNase I or shrimpnuclease) with reaction buffer (4 samples) to a total volume of 6 ul.Samples were incubated at room temperature for 5 minutes and then thenuclease was inactivated by heating to 85° C. for 5 minutes in thepresence of EDTA. Reverse transcription components were added to thesamples using the Transcriptor First Strand cDNA Synthesis kit (Roche)with a blend of oligo(dT) and random hexamers in total volume of 20 μl(20mM final GTC concentration per RT reaction). To further test theeffect of nuclease treatment samples were extracted and QPCR amplified,but without reverse transcription (NoRT controls). Hence, no RNA isconverted to cDNA and the QPCR measure the presence of the contaminatinggenomic DNA. All samples were assayed by QPCR for 18 S rRNA. ACTB andGAPDH using SYBR Green I chemistry in a LightCycler480 (Roche).

Example 9 Improved Reproducibility with the Disclosed Invention

Eight individual pancreatic β-cells were collected in either 0.5% NP-40or in 0.5 M GTC according to the disclosed invention. After lysis theRNA was split in three reverse transcription reactions (SuperScript III,Invitrogen), cDNA was quantified by QPCR in a 7900 Taqman instrumentfrom Applied Biosystems. The intra-assay variation, represented here bythe standard deviation (SD) of the three CT values obtained for each ofthe eight cells, is shown in FIG. 9. The SD based on the disclosedinvention was consistently lower demonstrating improved reproducibilityof the disclosed process.

Example 10 Comparison Between Current State of the Art Technology andthe Disclosed Invention.

Human cervical samples containing the human papilloma virus (HPV) wassplit into two aliquots of 300 μl each. The first aliquot was extractedusing the state of the art RNeasy Micro kit (QIAGEN) according to themanufacturer's protocol. The second aliquot was extracted using thedisclosed invention as follows. The aliquoted material was centrifugedfor 5 minutes at 2300g, washed with 500 μl PBS, centrifuged again for 3minutes at 2300g and the supernatant was discarded. 2 μl of GTCcontaining lysis buffer (0.2 M) according to the disclosed invention wasadded. The aliquots were vortexed for one minute, transferred to 0.2 mltubes and incubated at 80° C. for 5 minutes. Thereafter they were placedon ice.

Reverse transcription reagents were added in the form of theTranscriptor First Strand cDNA Synthesis kit (Roche) with a blend ofoligo(dT) and random hexamers in a total volume of 20 μl (final GTCconcentration was 0,2M). The presence of HPV16 was quantified with qPCRand FAM labelled TaqMan probes using the Eppendorf Realplcx system. ForHPV16E7 the difference in CT values with the two methods was37.2−29.2=8, corresponding to about 2̂ 8=256 times more material beingdetected with the disclosed invention. HPV16E6 was only detected whenthe sample was extracted with the disclosed invention.

Example 11 Removal of Deoxyribonucleic Acid in Cervical Scrapes Samplesas Integrated Step in the Disclosed Invention.

Caski cells with a total volume of 500 μl were extracted using thedisclosed invention and reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis kit (Roche) with a blend of oligo(dT) and randomhexamers. The sample was incubated at 80° C. for 5 minutes. A DNasespecific for double stranded DNA was added to the reverse transcriptionmix (a final concentration of 6 U shrimp DNase). Both the reversetranscription enzyme and the shrimp DNase were inactivated by heating to85° C. for 5 minutes. The sample was analyzed by QPCR using an EppendorfRealplex, Controls were run with reverse transcription and/or withoutDNase treatment. Treatment with DNase shifted CT values substantially tohigher values evidencing that the DNase step to remove contaminatinggenomic DNA was successfully integrated in the disclosed invention.

Example 12 Analysis of Pelleted Monocytes with the Disclosed InventionUsing Different Lysis Temperatures.

THP1 monocyte aliquots of approximately 35000 cells were pelleted bycentrifugation at 300g for 5min and washed with 2000 μl cold PBS. Afterremoval of PBS, 2μl of lysis buffer containing 0.2 M GTC and 20 ngpolyinosinic acid according to the present invention was added to eachwell. The samples were incubated at either 80° C. for 5 minutes or atroom temperature for 10 or 20 minutes. Reverse transcription (RT) wasperformed directly on the lysates in a 20 μl reaction volume (final GTCconcentration was 20 mM) using the Transcriptor First Strand cDNAsynthesis kit (Roche) with a blend of oligo(dT) and random hexamers. TwomRNAs. RRN18S and ACTB, were quantified in each aliquot with qPCR andSYBR Green I detection m a LightCycler480 (Roche).

LIST OF ABBREVIATIONS USED

-   Ct, Cycle of threshold-   GSP, gene specific RT-primer-   qRT-PCR, quantitative reverse transcription polymerase chain    reaction-   RT, reverse transcription-   SD, standard deviation

1. A method for performing a real time polymerase chain reaction(RT-PCR) for amplifying a target RNA comprising the steps of: lysing abiological sample which is supposed to contain said target RNA in asample vessel with a lysis buffer comprising between 0.05 M and 1 M ofGuanidine Thiocyanate, diluting said sample to an extent such that saidGuanidine Thiocyanate is present for a subsequent reverse transcriptionstep in a concentration of about 30 to 50 mM in said sample vessel,without any intermediate purification step, reverse transcribing saidtarget RNA in the presence of a mixture of first strand cDNA synthesisprimers into a first strand cDNA, said mixture consisting of primershybridizing to a poly-A sequence or random primers or target specificprimers in said sample vessel, and amplifying said first strand cDNA bymeans of subjecting said sample to multiple cycles of a thermocyclingprotocol.
 2. The method according to claim 1 wherein said amplificationis monitored in real time.
 3. The method according to claim 1 whereinsaid biological sample consists of not more than 1000 cells.
 4. Themethod according to claim 1 wherein said biological sample consists ofnot more than 100 cells.
 5. The method according to claim 1 wherein saidbiological sample consists of not more than a single cell.
 6. (canceled)7. The method according to claim 1 wherein said lysis buffer comprisesbetween about 0.2 and 0.5 M Guanidine Thiocyanate.
 8. (canceled)
 9. Themethod according to claim 1 wherein the lysis step is performed in thepresence of NP40 (octyl phenoxylpolyethoxylethanol) and wherein saidnon-ionic detergent during the reverse transcription step has a V/V of0.5 to 2%.
 10. The method according to claim 1 wherein the lysis stepcomprises the addition of a carbohydrate, which is preferably a sugar ora dextran.
 11. The method according to claim 1 wherein the lysis step isperformed for at least 5 minutes at ambient temperature or below ambienttemperature.
 12. The method according to claim 1 wherein the lysis stepis performed for at least 5 minutes at a temperature between about 55°C. to 85° C., preferably in the presence of proteinase K.
 13. The methodaccording to claim 12 wherein between the lysis step and the dilutionstep or between the dilution step and the reverse transcription step,the sample is incubated for at least 5 minutes at a temperature betweenabout 80° C. to 90° C.
 14. The method according to claim 11 wherein thelysis step is performed in the presence of DNAse I or Shrimp Nuclease.15. The method according to claim 14 wherein between the lysis step andthe dilution step or between the dilution step and the reversetranscription step, the sample is incubated for at least 5 minutes at atemperature between about 80° C. to 90° C.
 16. The method according toclaim 11 wherein prior to the dilution step, the sample is frozen attemperatures between about −20° C. and −80° C.
 17. The method accordingto claim 1 wherein said mixture of cDNA synthesis comprises primershybridizing to a poly-A sequence and random primers.
 18. The methodaccording to claim 17 wherein primers hybridizing to a poly-A sequenceand random primers are present in essentially equal molar amounts. 19.The method according to claim 17 wherein said primers hybridizing to apoly-A sequence and random primers are present in concentrations between1 μM and 5 μM each.
 20. The method according to claim 17 wherein saidprimers hybridizing to a poly-A sequence and random primers are presentin concentrations of about 2.5 μM each.